The formation of boundaries between or within tissues is a fundamental aspect of animal development. Boundaries function to separate populations of cells with different identities, allowing them to follow independent developmental programs in spite of their close proximity. Additionally, boundaries can form important organizing centers that pattern adjacent cells. In the developing vertebrate central nervous system, boundaries form in an apparently homogeneous neuroepithelium, separating brain regions that subsequently acquire distinct histological and functional properties. In the hindbrain in particular, boundaries form between the rhombomeres, producing a series of 7 segments along the main body axis. In spite of the fundamental importance of boundaries in animal development, surprisingly little is known about the molecules and mechanisms that control boundary formation. Even in Drosophila, where compartment boundaries have intrigued researchers for decades, the actual molecules that mediate boundary formation have only recently begun to be elucidated. The long-term goal of the proposed research is to understand the cellular and molecular basis of rhombomere boundary formation and maintenance during vertebrate hindbrain development. We understand that the appearance of a sharp developmental boundary involves 2 steps: first, long-range signals establish broad domains with distinct regional identities but diffuse boundaries, and secondly fine-scale interactions between cells at the edges of these domains result in the sharpening of the boundaries. In Aims 1 and 2 of this proposal we investigate the mechanisms underlying rhombomere boundary sharpening by studying two parallel processes: cell sorting, whereby cells on the "wrong" side of a forming boundary move to the "right" side, and cell plasticity, whereby cells on the "wrong" side change their identity to match that of their surroundings. Aim 1 addresses the mechanism of cell sorting, and tests the hypothesis that sorting occurs in response to 2 parallel influences: cell-cell repulsion between unlike cells at rhombomere boundaries, and cell-cell adhesion between like cells within rhombomeres. Aim 2 addresses the mechanism of plasticity. Finally, in Aim 3, we consider the mechanism by which long-range signals interact to specify the positions of specific hindbrain boundaries.